Challenging the status quo: a case for the lack of gut microbiota exacerbating hypertensive end-organ damage
Authors: Chudan Xu, Francine Z Marques
Journal: Cardiovascular Research (2023)

In the current issue of Cardiovascular Research, Avery et al. 1 sought to comprehensively investigate the impact of microbial colonization on end-organ damage in hypertension by using a powerful experimental model: germfree (GF) mice.Here, we highlight their novel findings and relevance to the field, particularly in how these findings challenge the status quo.Imbalances in the gut microbiome have been associated with the progression of experimental and clinical hypertension and its associated end-organ damage. 2 However, the gut microbiome is shaped by a broad variety of extrinsic (e.g.diet, birth mode) and intrinsic factors (e.g.host's genetics, gut transit time), many not considered in the design of microbiome studies. 2 Thus, studies that differentiate between association and causation are critical.In this setting, GF mice are one of the most powerful tools to study the role of the microbiome in a phenotype and to move the field from association to causation.Nonetheless, the use of GF mice is scarce as they are costly and technically challenging to maintain and surgically manipulate, limiting the number of studies using them as a model.Therefore, to exclude confounding effects, Avery et al. 1 utilized a sophisticated study design to quantify the impact of microbial colonization in angiotensin II-induced hypertension: they randomized GF littermate mice and microbially colonized half of them as controls.They did so by placing C57BL/6J GF mice in cages that were previously used by healthy male mice of the same strain in a specific pathogen-free facility for 8 weeks prior to surgery. 1 Using this approach, the authors demonstrated that GF mice had slightly higher mean arterial pressure at baseline (albeit still within a 'normotensive' range), with a similar vascular reactivity to angiotensin II, but stronger adverse cardiorenal responses to hypertension than their conventionalized GF counterparts. 1Importantly, GF mice developed a particularly exacerbated renal damage, with more fibrosis, higher albumin-to-creatinine ratio, and increased infiltration of inflammatory immune cells (CD4+ and CD8+ T cells and F4/80+ macrophages) relative to conventionalized GF mice (Figure 1). 1 Controversially, previous findings from Karbach et al. 3 showed that GF mice were protected from developing hypertension and its related inflammatory stress and vascular damage.There are obvious differences in study design that might underlie the conflicting findings (Figure 1).The first and most important is that the use of conventionalized GF littermate mice as control, as performed by Avery et al., 1 is ideal, as this minimizes several factors that affect the microbiome, including genetic drifts and in utero delivery of gut microbial metabolites.Moreover, there were differences in protocol length (7 vs. 14 days of angiotensin II), use of sodium in the drinking water, and angiotensin II dose (1 mg/kg/day vs. 1.44 mg/kg/day).Less obvious, but equally important, differences that affect the gut microbiome include diet, the facility and housing conditions used-these would have an important impact on the gut microbiome and associated derived metabolites that could impact the hypertensive response to angiotensin II.For example, a microbiota resultant of low fibre intake could have exacerbated the hypertensive response 4 observed by Karbach et al. 3 Thus, the findings by Avery et al. emphasize the recent position paper by the European Society of Cardiology (ESC) Working Group on Coronary Pathophysiology and Microcirculation, 5 including open-access microbiome data and detailed experimental protocols, that can improve the reproducibility and translation of findings in hypertension research.This highlights the need for the use of guidelines (such as Marques et al. 6 ) in gut microbiome studies.Both studies, however, were performed only in male mice, highlighting an important gap in hypertensive gut microbiome research: sex differences. The interplay of the gut microbiota and their metabolites with the immune systemSome of the origins of an exaggerated pro-inflammatory immune response in hypertension are still unknown.A key link may be the gut microbiome and microbial-derived metabolites, such as short-chain fatty acids (SCFAs), which reduced BP in mice and hypertensive patients 2,4,7 and were reduced in GF mice. 1 The lower/absent levels of SCFAs may explain, at least partially, the cardiorenal phenotype observed in hypertensive GF mice. 1 Avery et al. demonstrated that, in the absence of microbes and their metabolites, naïve T cells from GF mice were more prone to differentiate into T-helper 17 (T H 17) cells in vitro, which yielded higher pro-inflammatory cytokines expression in response to angiotensin II. 1 Considering their earlier findings that sodium increased the number of T H 17 cells 8 and that, in the present study, only angiotensin II-treated mice received sodium in their drinking water, 1 it is plausible to hypothesize that this effect could be sodium-driven.Importantly, pre-treatment with SCFAs on GF-derived cells ameliorated the observed inflammation signs in response to angiotensin II. 1 Avery et al. proposed SCFAs could prime immune cells and allow them to be more prepared to become anti-